Mild hypothermia is the most effective therapy against experimental ischemic cerebral damage that currently exists. Mild hypothermia is already being successfully used in the treatment of acute traumatic brain injury and the feasibility of using it to treat stroke patients is currently being evaluated in some clinical trials. While multiple mechanisms for hypothermia-induced neuroprotection have been suggested, these mechanisms remain unclear and several key issues still need to be addressed. This project using a fresh approach using molecular biology techniques to test the hypothesis that mild hypothermia's neuroprotective benefit is due in part to a) attenuation of deleterious reactive oxygen species (ROS) and b) to inhibition of apoptotic cell death. Models of focal and global cerebral ischemia will be used and mild hypothermia will be applied to intraischemically and in a delayed fashion to mimic clinically relevant paradigms. A series of experiments will systemically examine the effects of mild hypothermia on 1) the generation of ROS in various brain regions during early reperfusion and at the peak of the inflammatory process; 2) the expression of endogenous antioxidants such as superoxide dismutases (SOD1 and SOD2), and glutathione peroxidase; 3) the expression of a death promoting (Bax) and death suppressing gene (Bcl-2); 4) the release of cytochrome c and caspase expression (ICE, CPP32); 5) DNA fragmentation characteristic of programmed cell death. This proposal will further test whether mild hypothermia can attenuate the increased neuronal damage observed in SOD-deficient mice and whether it does so by reducing ROS production, by altering the cytosolic translocation of cytochrome c, and/or by altering the regulation of Bcl-2 and Bax. The ability of post-ischemic administration of an antioxidant to extend the therapeutic window of mild hypothermia will also be tested. The knowledge gained from this project will elucidate novel cellular and molecular mechanisms underlying mild hypothermia's neuroprotective benefit, contribute to understanding the pathophysiology of ischemic cerebral injury, and may have important implications for the treatment of clinical stroke in humans.